U.S. patent number 4,705,848 [Application Number 06/869,702] was granted by the patent office on 1987-11-10 for isolation of bioactive, monomeric growth hormone.
This patent grant is currently assigned to International Minerals & Chemical Corp.. Invention is credited to Edwin J. Hamilton, Jr., Larry D. Taber, Ren-Der Yang.
United States Patent |
4,705,848 |
Yang , et al. |
November 10, 1987 |
**Please see images for:
( Certificate of Correction ) ** |
Isolation of bioactive, monomeric growth hormone
Abstract
Monomeric, biologically active growth hormone is isolated from
microbially-produced insoluble inclusion bodies by solubilizing and
denaturing the growth hormone by extraction of the inclusion bodies
into a guanidine salt solution such as guanidine hydrochloride and
subsequently renaturing at least a portion of the growth hormone in
the solution by replacing the guanidine salt solution with a
denaturant-free buffer solution and removing precipitated
impurities and growth hormone aggregates. The renatured growth
hromone is then purified by ion-exchange chromatography.
Inventors: |
Yang; Ren-Der (Terre Haute,
IN), Hamilton, Jr.; Edwin J. (Terre Haute, IN), Taber;
Larry D. (Indianapolis, IN) |
Assignee: |
International Minerals &
Chemical Corp. (Terre Haute, IN)
|
Family
ID: |
25354104 |
Appl.
No.: |
06/869,702 |
Filed: |
June 2, 1986 |
Current U.S.
Class: |
530/399; 530/397;
530/808; 530/825 |
Current CPC
Class: |
C07K
1/1136 (20130101); C07K 14/61 (20130101); Y10S
530/808 (20130101); Y10S 530/825 (20130101) |
Current International
Class: |
C07K
1/00 (20060101); C07K 1/113 (20060101); C07K
14/435 (20060101); C07K 14/61 (20060101); A61K
037/36 () |
Field of
Search: |
;530/399,397,808,825 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0116778 |
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Jun 1984 |
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EP |
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0114507 |
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Aug 1984 |
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EP |
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0122080 |
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Oct 1984 |
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EP |
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0121775 |
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Oct 1984 |
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EP |
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0123928 |
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Nov 1984 |
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EP |
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WO83/04418 |
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Dec 1983 |
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WO |
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WO84/03711 |
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Sep 1984 |
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WO |
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2129810 |
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May 1984 |
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GB |
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Primary Examiner: Schain; Howard E.
Attorney, Agent or Firm: Guffey; Wendell R. Shearer; Peter
R. Farquer; Thomas L.
Claims
What is claimed is:
1. A process for recovering and purifying monomeric, biologically
active growth hormone from insoluble inclusion bodies produced by
expression of a heterologous gene in a microorganism, which process
comprises:
(a) solubilizing and denaturing the growth hormone by extracting
the inclusion bodies into a solution of a guanidine salt;
(b) renaturing at least a portion of the growth hormone in the
solution and inducing precipitation of at least a portion of
contaminant proteins and aggregates in the solution by replacing
the guanidine salt solution with a denaturant-free buffer solution
without the use of an intermediate denaturant, thereby reducing the
presence of soluble contaminant proteins and agregates in the
renatured protein solution which tend to foul the chromatography
column and which are not biologically active;
(c) removing the precipitated contaminants and aggregates from the
denaturant-free buffer solution; and
(d) purifying the monomeric growth hormone in the solution by ion
exchange chromatography.
2. A process as claimed in claim 1, wherein the guanidine salt is
guanidine hydrochloride.
3. A process as claimed in claim 2, wherein the guanidine
hydrochloride solution has a concentration from about 6 to about 8
M.
4. A process as claimed in claim 3, wherein the guanidine
hydrochloride solution also contains ethanolamine at a
concentration from about 20 mM to 100 mM.
5. A process as claimed in claim 4, wherein the inclusion bodies
are dissoIved in the guanidine hydrochloride solution in an amount
sufficient to give a growth hormone concentration of from about 1
to about 2.5 grams per liter.
6. A process as claimed in claim 2, wherein the solution of growth
hormone in guanidine hydrochloride is allowed to stand for a period
of from about 6 to about 36 hours prior to renaturation.
7. A process as claimed in claim 2, wherein the guanidine salt is
removed by diafiltration or dialysis.
8. A process as claimed in claim 2, wherein the guanidine salt is
removed over a period of less than about 10 hours.
9. A process as claimed in claim 2, wherein the growth hormone is
porcine growth hormone or a biologically active fragment or analog
thereof.
10. A process as claimed in claim 2, wherein the growth hormone is
bovine growth hormone or a biologically active fragment or analog
thereof.
11. A process as claimed in claim 2, wherein, prior to solubilizing
the inclusion bodies, the inclusion bodies are washed in a buffered
solution which is devoid of detergents, reducing agents and
enzymes.
12. A process as claimed in claim 11, wherein the solution in which
the inclusion bodies are washed contains ethylenediaminetetraacetic
acid and monosodium phosphate.
13. A process for recovering and purifying monomeric, biologically
active porcine growth hormone, bovine growth hormone, or
biologically active fragments or analogs thereof from insoluble
inclusion bodies produced by expression of a heterologous gene in a
microorganism, which process consists essentially of:
(a) washing the inclusion bodies in a buffered solution which is
devoid of detergents, reducing agents and enzymes;
(b) solubilizing and denaturing the growth hormone by extracting
the inclusion bodies into about a 6 to 8 M solution of a guanidine
hydrochloride;
(c) renaturing at least a portion of the growth hormone in the
solution and inducing precipitation of at least a portion of
contaminant proteins and aggregates in the solution by replacing
the guanidine hydrochloride solution with a denaturant-free buffer
solution without the use of an intermediate denaturant, thereby
reducing the presence of soluble contaminant proteins and
aggregates in the renatured protein solution which tend to foul the
chromatography column and which are not biologically active;
(d) removing the precipitated contaminants and aggregates from the
denaturant-free buffer solution; and
(e) purifying the monomeric growth hormone in the solution by ion
exchange chromatography.
14. A process as claimed in claim 13, wherein the guanidine
hydrochloride solution also contains ethanolamine at a
concentration from about 20 mM to 100 mM.
15. A process as claimed in claim 14, wherein the inclusion bodies
are dissolved in the guanidine hydrochloride solution in an amount
sufficient to give a growth hormone concentration of from about 1
to about 2.5 grams per liter.
16. A process as claimed in claim 13, wherein the solution of
growth hormone in guanidine hydrochloride is allowed to stand for a
period of from about 6 to about 36 hours prior to renaturation.
17. A process as claimed in claim 13, wherein the guanidine
hydrochloride is removed by diafiltration or dialysis.
18. A process as claimed in claim 13, wherein the guanidine
hydrochloride is removed over a period of less than about 10
hours.
19. A process as claimed in claim 13 wherein the solution in which
the inclusion bodies are washed contains ethylenediaminetetraacetic
acid and monosodium phosphate.
Description
BACKGROUND OF THE INVENTION
The advent of recombinant DNA technology has made possible the
large scale production of proteins by the insertion of
heterologous, protein-encoding genes into microorganisms such as
bacteria and expression of the genes within the host
microorganisms. In this manner, a large variety of proteins,
including some which can only be obtained in minute quantities from
natural sources, can be economically produced in unlimited
quantities.
Unfortunately, proteins which are native to eukaryotic cells may
not undergo post-translational processing in microbial hosts to
yield biologically active forms of the desired proteins. Thus, for
example, eukaryotic proteins containing multiple cysteine residues
may not form the correct disulfide linkages necessary for
biological activity when they are expressed in microbial hosts. Not
only may the eukaryotic protein fold improperly within the
intracellular environment of the host, but also the individual
molecules may form biologically inactive aggregates or oligomers as
the result of the formation of intermolecular disulfide bonds or
other types of intermolecular bonding.
As the result of one or more of these phenomena--improper folding,
incorrect disulfide bond formation and non-covalent or covalent
oligomerization--many proteins that are produced by the expression
of heterologous genes in microbial hosts are not recovered from the
host cells in the form of soluble, biologically active protein.
Rather, upon lysis of the cells, the heterologous proteins are
found in the form of insoluble "inclusion bodies," also sometimes
referred to as "refractile bodies." In order to produce useful
proteins, a means must be provided whereby the inclusion body
proteins can be converted into a monomeric, biologically active
form in which they are soluble in biological fluids.
In addition to converting the inclusion body proteins into soluble,
monomeric, biologically active forms, it is necessary at some point
in the recovery process to purify the protein in order to remove
bacterial impurities including endotoxins, other bacterial proteins
and contaminating substances derived from the bacterial host and/or
the fermentation medium. This is usually done by subjecting the
protein to some of the various chromatographic purification
procedures such as ion-exchange chromatography.
PCT Application No. GB 83/00152 discloses methods for recovering
and activating the milk-clotting enzyme chymosin, beginning with
inclusion bodies produced in E. coli which contain the enzyme in
its zymogenic form. The methods involve dissolving the inclusion
body protein in denaturants such as urea, guanidine hydrochloride
or alkali solution, renaturing the protein by removing or diluting
the denaturant and reducing the pH of the solutions to induce
autocatalytic cleavage of the zymogen to the mature form of the
protein.
Solubility and folding characteristics vary considerably between
different proteins, since both are highly dependent on the primary
structure, i.e., amino acid sequence, of the protein. It has been
the experience of the prior art that animal growth hormones are
particularly difficult proteins to recover in soluble, monomeric,
biologically active form. Thus, for example, it is said in U.S.
Pat. No. 4,512,922 that, for proteins such as growth hormones,
dissolution of the inclusion body protein in a strong denaturant
followed by dilution of the denaturant with aqueous buffer almost
invariably results in reprecipitation of the protein. Even if
reprecipitation does not occur, expected levels of activity are
said not to be shown. As a solution to this problem, there is
disclosed a method for purifying growth hormone in which the
inclusion body proteins are solubilized in a strong denaturant; the
strong denaturant is replaced by a weaker denaturant; and the
weaker denaturant is subsequently removed to renature the
protein.
We have found that a two-stage renaturation process, such as that
disclosed in U.S. Pat. No. 4,512,922, entails a number of problems.
The yields of soluble, biologically active growth hormone
obtainable are not particularly good. Moreover, the method yields
varying results depending on the species of growth hormone
involved. For example, using 8 M guanidine hydrochloride as a
strong denaturant and 3.5 M urea as the weaker denaturant, we have
found that yields of soluble, biologically active porcine growth
hormone were only on the order of about 1% or less. While bovine
growth hormone yields were somewhat higher, on the order of about
5%, these were still only marginal from a commercial point of view.
Moreover, problems arose in the purification of the proteins
recovered by this process. When the proteins recovered in this
manner were loaded onto an ion-exchange column for purification,
large quantities of soluble protein aggregates bound to the column,
causing it to become fouled and obstructed within a relatively
short period of time. This was true even when the column
purification was carried out under reducing conditions in an
attempt to eliminate aggregates. The use of the two-stage
renaturation process is also problematical from a commercial
production standpoint inasmuch as it entails numerous processing
steps and expensive reagents.
We have also attempted to recover growth hormones from inclusion
bodies by solubilizing the inclusion body proteins in 8 M urea and
subsequently renaturing the protein in a single step by dialysing
the solution against denaturant-free buffer to remove the urea.
Yields of recovered monomeric growth hormone were very poor, that
is, on the order of 1% or less.
It is an object of this invention to provide an efficient method
for recovering microbially produced growth hormone in a soluble,
monomeric, biologically active form.
It is a further object of the invention to provide a method for
recovering and purifying microbially produced growth hormone using
a chromatographic purification column whereby the purification
column does not become plugged and obstructed within a short period
of time.
Other objects and advantages of the invention will be readily
apparent from the description of the invention which follows.
SUMMARY OF THE INVENTION
This invention provides an efficient, economical method for
isolating and purifying soluble, bioactive, monomeric growth
hormone from inclusion bodies. The method of the invention avoids
many of the problems associated with the two-stage renaturation or
urea-based methods of the prior art. In particular, the process of
the invention substantially reduces the presence of soluble GH
aggregates and thus alleviates the problem of column fouling during
ion exchange chromatography. Furthermore, the process of the
invention reduces the number and cost of reagents used in the
recovery process.
Contrary to the teachings of the prior art, we have found that
under appropriate conditions, growth hormone inclusion bodies can
be solubilized in a guanidine salt solution and thereafter
renatured by a single-step removal of guanidine. When guanidine is
removed in a rapid single-step renaturation, essentially all
protein aggregates precipitate from the solution, allowing them to
be separated from soluble, monomeric growth hormone prior to
chromatographic purification. Consequently, fouling and obstruction
of the column is minimized and the useful life of the column is
extended.
In the practice of the invention, the inclusion body is solubilized
and the protein denatured by extracting it into an aqueous solution
of a guanidine salt preferably guanidine hydrochloride. The
guanidine salt solution is then replaced by denaturant-free buffer
solution, for example, by dialysis, causing at least a portion of
the denatured growth hormone to refold to its monomeric, native
configuration. Concomitantly, some proteinaceous contaminants as
well as almost all growth hormone aggregates which may be present
in the solution are precipitated. The precipitated contaminants and
aggregates are easily separated and the remaining solution of
monomeric growth hormone, in its biologically active form, is then
further purified by ion exchange chromatography. The replacement of
guanidine salt with denaturant-free buffer solution is carried out
without the use of an intermediate denaturant. In a commercial
scale production process, elimination of the intermediate
denaturant represents a substantial savings in material costs.
DETAILED DESCRIPTION OF THE INVENTION
The process of the invention can be employed in the isolation of
monomeric, biologically active forms of any animal growth hormones
which are found as insoluble inclusion bodies in microorganisms,
such as bacteria, particularly bovine growth hormone (bGH) or
porcine growth hormone (pGH), including biologically active
fragments thereof and analogs which have differences in amino acid
sequence but still display growth hormone activity.
Methods for preparing expression vectors capable of expressing
growth hormone in bacterial hosts are known in the art (see, e.g.,
Seeburg et al., DNA, 2:37-45 [1983] and Goeddel et al., Nature,
281:544-548 [1979]). In one embodiment of the process of the
invention, we used pGH-containing inclusion bodies which were
produced by an E. coli host strain HB101 transformed with a first
plasmid, pL-mu-.DELTA.7 SGH, coding for .DELTA.7 pGH (porcine
growth hormone less its seven N-terminal amino acids plus
methionine and serine) under the control of a phage lambda promoter
and a second plasmid, pCI857, which codes for the
temperature-sensitive lambda phage repressor protein. In another
embodiment, we employed bGH-containing inclusion bodies which were
produced by an E. coli host strain HB101 transformed with a
plasmid, pL- mu-.DELTA.9 bGH, coding for .DELTA.9 bGH (bovine
growth hormone less its nine N-terminal amino acids plus methionine
and serine) and with plasmid pCI857. It will be readily apparent,
however, that the process of this invention is equally applicable
to the purification of recombinant growth hormone produced by any
host/vector combination, provided only that the growth hormone is
produced in the host in the form of an insoluble inclusion
body.
Prior to using the recovery and purification procedure of the
invention, the transformant cells are generally lysed, either
mechanically or enzymatically, to allow recovery of the inclusion
bodies which are sequestered within the cells. The inclusion bodies
can be separated from the bulk of the remainder of the cellular
material by centrifugation and washing in a buffer. Preferably, the
cell paste which is obtained by separating the cells from the
fermentation medium is dispersed in an aqueous buffer solution
containing ethylenediaminetetraacetic acid (EDTA) (20 millimolar)
and monosodium phosphate (100 millimolar) adjusted with sodium
hydroxide to pH 7.8. The cells are disrupted to release the growth
hormone inclusion bodies by passage one or more times through a
poppet-type homogenizer such as a Manton-Gaulin homogenizer. Growth
hormone pellets are separated from the solution substrate and from
some of the cell debris matter by centrifugation. The resulting
pellets are washed one or more times by resuspension in an
EDTA-monosodium phosphate buffer solution (10 millimolar EDTA, 0.2
molar NaH.sub.2 PO.sub.4, adjusted to pH 7.5) and are separated
from the wash solution by centrifugation. The resulting washed
pellets are stored at about 4.degree. C. if they are to be used
within a few days or they are frozen if they are to be used at a
later date.
The recovery procedures taught by the prior art generally employ
wash steps that use detergents such as Triton X-100, reducing
agents such as 2-mercaptoethanol and enzymes such as lysozyme in
order to maximize the removal of cell debris and contaminating
protein at the wash stage. We have found, however, that the use of
such agents is unnecessary in the wash step. Moreover, their
exclusion from the wash step is advantageous inasmuch as they cause
some of the desired protein to be lost in the wash. Even though
elimination of these agents from the wash allows some additional
contaminants to be carried through to the solubilization step, we
have found that some of these contaminants are precipitated later
in the process without the use of any additional reagents.
The inclusion bodies containing the growth hormone are solubilized
and the protein is denatured by extraction into an aqueous solution
of a guanidine salt, preferably guanidine hydrochloride. Guanidine
hydrochloride, which is a strong chaotrope, is capable of
completely, but reversibly, denaturing proteins at concentrations
of 6-8M. Advantageously, the guanidine hydrochloride solution is
purified prior to use in the process of the invention in order to
remove high molecular weight impurities which may be present. The
impurities may be removed by ultrafiltration or by any other
suitable purification means known in the art. Preferably, the
solution also contains ethanolamine at a concentration of from
about 20 mM to 100 mM. The inclusion bodies preferably are
dissolved in the guanidine hydrochloride solution in an amount
sufficient to give a concentration of growth hormone of from about
1 to about 2.5 grams per liter of solution. The solution is then
allowed to stand for a sufficient amount of time to allow the
completion of molecular unfolding. We have found that a period of
from about 6 to about 36 hours is satisfactory.
After the growth hormone has been solubilized and denatured, at
least a portion of the growth hormone in the solution is renatured
by replacing the guanidine hydrochloride with denaturant-free
buffer solution. Whereas the prior art teaches the slow removal of
denaturant in order to maximize the amount of protein which remains
in solution, we have found that a fairly rapid removal of guanidine
salt is actually preferred because it reduces the presence of
soluble protein aggregates in the renatured protein solution which
tend to foul the chromatography column and which are not
biologically active. Consequently, we prefer to remove the
guanidine salt over a period of less than about 10 hrs. The ability
to remove substantially all of the aggregated growth hormone as
precipitate prior to ion-exchange chromatography is a major
advantage of our method.
Replacement of the guanidine hydrochloride can be effected by any
of the known methods for removing small molecules from protein
solutions. Preferred methods for removing the guanidine
hydrochloride include diafiltration and dialysis. We employ a
hollow fiber ultrafiltration unit such as a Romicon HF4S, which is
commercially available from Romicon, Inc. This unit employs a
hollow fiber ultrafiltration membrane which allows the passage of
molecules having molecular weights below about 10,000. The
circulation of growth hormone solution past the ultrafiltration
membrane results in the passage of guanidine hydrochloride solution
through the membrane while the growth hormone is retained. The
volume of solution is maintained by feeding a diluent solution
containing ethanolamine at a concentration of about 60 millimolar
adjusted to a pH of 9.0 to 9.8. The amount of diluent feed is about
5 to about 7 volumes per volume of growth hormone solution and the
rate of feed is about 0.5 to about 4 volumes per hour. Accordingly,
the flow rate of liquid amounts to about 120 liters per hour over
100 sq. ft. of membrane surface in the hollow fiber ultrafiltration
unit.
Alternatively, the guanidine-containing solution can be diluted
until the concentration of guanidine salt is so low that the growth
hormone undergoes renaturation. This occurs at guanidine
concentrations below about 1M.
As the guanidine hydrochloride is removed from the solution, some
proteinaceous contaminants and growth hormone aggregates which are
present precipitate from the solution. The precipitates can be
removed from the solution by known methods such as centrifugation
or filtration. If desired, the growth hormone solution may be
concentrated somewhat by ultrafiltration prior to the
centrifugation step.
The solution containing the soluble, monomeric growth hormone is
purified by ion exchange chromatography. The ion exchange
chromatography employs conventional equipment, such as a Pharmacia
ion exchange column, which employs a DE-52 Cellulose ion exchange
resin. The solution is loaded onto the column and the purified,
bioactive growth hormone is collected in the run-through fraction
which does not bind to the column.
Following ion exchange chromatography, the recovered growth hormone
can be subjected to any conventional processing steps such as
concentration by ultrafiltration, additional purification steps
and, if desired, lyophilization to produce the growth hormone in a
stable powdered form.
The following examples are intended to further illustrate the
practice of the invention. Unless otherwise indicated, all percents
are by weight and all temperatures are in degrees C.
EXAMPLE I
The transformant cells containing inclusion bodies of porcine
growth hormone (pGH) that were used in this example were produced
as follows:
Samples of E. coli HB101 (P.sub.L -mu-.DELTA.7 SGH and pcI857)
cells, ATCC 53031, to which 10% (v/v) glycerol had been added, were
stored under liquid nitrogen or at -85.degree. C. until needed.
A. Inoculation
The inoculum for a 9-liter fermentor charge was obtained by adding
the cells to 200 ml of either ESM-1 or ESM-2 medium contained in a
500 ml flask. The pH of the medium was adjusted to a value of 7.0.
The flask was closed with two milk filters so that some aeration of
the medium could take place while the flask was shaken at 300 rpm
for 16-20 hours at 30.degree. C. in a New Brunswick Rotary
Shaker.
______________________________________ Ingredient ESM-1 ESM-2
______________________________________ NZ Amine A 16 g/L 23 g/L
Glycerol 30 30 KH.sub.2 PO.sub.4 5 (NH.sub.4).sub.2 HPO.sub.4 2.5
MgSO.sub.4.7H.sub.2 O 7 7 K.sub.2 HPO.sub.4 6 (NH.sub.4).sub.2
SO.sub.4 5 NaH.sub.2 PO.sub.4 3 Na Citrate 1 Trace Element
Solution* 20 ml 20 ml ______________________________________ *Trace
element solution G/L: EDTA 5, FeCl.sub.3.6H.sub.2 O 0.5, ZnO 0.05,
CuCl.sub.2.2H.sub.2 O 0.01, Co(NO.sub.3).sub.2.6H.sub.2 O 0.01,
(NH.sub.4).sub.2 MoO.sub.4 0.01.
B. Fermentor
The fermentor was a New Brunswick Microgen with a total volume of
16 liters. Nine liters of liquid medium was initially charged to
the fermentor plus 180 ml of inoculum.
C. Fermentation Medium
The composition of the initial 9-liters of medium is shown
below:
______________________________________ Concentration Grams Product
per 9 Liters ______________________________________ NZ Amine
A-Sheffield 250 Glycerol 500 (NH.sub.4).sub.2 SO.sub.4 50 K.sub.2
HPO.sub.4 60 NaH.sub.2 PO.sub.4 30 Na Citrate 10
MgSO.sub.4.7H.sub.2 O 70 Hodag K-67 antifoam 4 ml
FeCl.sub.3.6H.sub.2 O 0.1 g ZnO 0.01 g CuCl.sub.2.2H.sub.2 O 0.002
Co(NO.sub.3).sub.2.6H.sub.2 O 0.002 (NH.sub.4).sub.2 Mo O.sub.4
0.002 EDTA (disodium salt) 1.0
______________________________________
The medium was sterilized for 20 minutes at 121.degree. C. and the
pH was adjusted to 6.8 with NaOH.
To the medium 250 mg, each of ampicillin and kanamycin were added.
The solution of antibiotics was sterilized by filtration.
D. Nutrient Feedings
At the time of induction, (i.e., when the temperature was raised to
42.degree. C.), nutrients were added to the fermentation medium 250
g NZ Amine A (enzymatic casein hydrolyzate) and 200 g glycerol in
approximately one liter water, were added. An additional feeding of
100 g NZ Amine A and 100 g of glycerol was given 5 hours
post-induction.
E. Fermentor Operation
The operating conditions that gave us our best results are set
forth in this section.
1. Growth Period 16-24 Hours
a. Temperature of medium=28.degree.-30.degree. C.
b. Agitator speed: 1000 RPM.
c. Energy input by the agitator 1.0-2.0 horsepower per 100
gallons.
d. Aeration rate: 10 L (STP) per minute.
e. Back pressure 5 lbs per in.sup.2.
f. Dissolved oxygen: Above 20% of air saturation value.
g. Absorbance of light (wavelength 550 nm) by the fermenting
medium. A.sub.550.
2. Induction Period
a. Temperature of medium.
(1) 42.degree. C. for the first hour of induction.
(2) 40.degree. C. for remainder of induction period.
b. Agitator speed: 1200 RPM.
c. Energy input by agitator: 0.5-1.5 horsepower per 100
gallons.
d. Aeration rate 10 L (STP) per minute.
e. Back pressure: 3-6 lbs per in.sup.2.
f. Dissolved oxygen: preferably above 20% of air saturation. In
order to obtain these values, the inlet air is enriched with
oxygen.
g. Final absorbance: A.sub.550 of 118-153.
Recovery of .DELTA.7-pGH from E. coli Cells
The cells obtained from 200 liters of fermentation broth produced
in a pilot plant by procedures described above for the 10-liter
fermentor were separated from the broth by centrifugation and
resuspended in 50 liters of a buffer containing EDTA (20 mM) and
NaH.sub.2 PO.sub.4 (100 mM), adjusted to pH 7.8 with sodium
hydroxide. The cell suspension was passed through a Manton-Gaulin
homogenizer two to three passes at a pressure of 8,000 psig in
order to disrupt the cells. Intact inclusion bodies of .DELTA.7-pGH
were collected by centrifugation (13,000 g, 10 minutes) and thus
separated from cellular debris. The recovered inclusion bodies
(7,000 grams) were then washed in a buffer containing EDTA (10 mM),
and a NaH.sub.2 PO.sub.4 (0.2 M) adjusted to pH 7.5 with sodium
hydroxide. The inclusion bodies were recovered from the washing
buffer by centrifugation and dissolved in 460 liters of 8 M
guanidine hydrochloride and 60 mM ethanolamine adjusted to pH 9.0
with sodium hydroxide. The solution was stirred for 12 hours to
complete the unfolding of the pGH molecules.
Guanidine hydrochloride was removed from the solution by
diafiltration through PM-10 membranes in the form of hollow fibers.
The membranes had an average pore size of 15 .ANG. which allows
passage of molecules having molecular weights of 10,000 or less.
After nearly all the guanidine hydrochloride had been removed from
the solution, the solution was centrifuged at 13,000 g for 10
minutes in order to remove proteinaceous impurities and pGH
molecular aggregates which precipitated out of the solution upon
removal of the guanidine hydrochloride. The pGH was then purified
by ion exchange chromatography using a Whatman DE-52 ion exchange
gel (DEAE Cellulose) loaded into a 25 centimeter by 15 centimeter
column. The solution containing the soluble, renatured pGH was
loaded onto the column and the pGH was collected in the run through
effluent which did not bind to the column. Column fouling and
plugging, which had been observed during ion exchange
chromatography when a similar urea-based recovery process was
employed, was not apparent. The ion exchange chromatography step
was repeated if the desired purity was not achieved.
The solution containing the pGH was then further purified by
ultrafiltration through a PM-10 hollow fiber membrane to yield a
solution containing 0.2% pGH. Low molecular weight contaminants
were then removed by ultrafiltration against Cornell buffers. This
procedure was done a first time against 50% Cornell buffer
(Na.sub.2 CO.sub.3, 11 mM; NaHCO.sub.3, 13 mM) and a second time
against 2% Cornell buffer (Na.sub.2 CO.sub.3, 0.42 mM; NaHCO.sub.3,
0.50 mM). The solution was then concentrated by ultrafiltration
through PM-10 hollow fibers to yield a solution containing 0.2% to
2% pGH. The solution was then centrifuged and the supernatant was
filtered through a 0.2 micron pore filter. The pGH in the solution
was then lyophilized to produce pGH in a powdered, bioactive
form.
EXAMPLE II
This example deals with the production of bovine growth hormone
(bGH). The transformant cells used for this example were made by
the following method.
Sample of E. coli HB101 (pL-mu-.DELTA.9 bGH and pCI857) cells, ATCC
53030, to which 4% (v/v) glycerol had been added, were stored under
liquid nitrogen until needed.
The inoculum for a 9-liter fermentor charge was obtained by adding
the cells to duplicate 500 ml baffled flasks each containing 200 mL
of LB medium. The LB medium had the following composition: 10 g per
L tryptone, 5 g per L yeast extract, 10 g per liter NaCl, 100 ug/ml
ampicillin plus 50 ug/ml kanamycin. The pH of the medium was
adjusted to a value of 7.0. The flasks were closed with a milk
filter closure so that some aeration of the medium could take place
while the flasks were shaken at 200 rpm for 15-20 hours at
30.degree. C. in a New Brunswick shaker.
The fermentor was a New Brunswick Microgen with a total volume of
16 liters. Nine liters of liquid medium were initially charged to
the fermentor plus 400 ml of inoculum. A.sub.550 of
inoculum=4-6.
A. Fermentation Medium
The composition of the initial 9 liters of medium is shown
below:
______________________________________ Product Concentration
Grams/Liter ______________________________________ NZ Amine
A-Sheffield 33.0 Glycerol 55.0 (NH.sub.4).sub.2 SO.sub.4 5.6
K.sub.2 HPO.sub.4 6.7 NaH.sub.2 PO.sub.4 3.3 Na Citrate 1.1
MgSO.sub.4.7H.sub.2 O 7.8 Hodag K-67 Antifoam 5 ml
FeCl.sub.3.6H.sub.2 O 0.014 ZnO 0.0014 CuCl.sub.2.2H.sub.2 O
0.00028 Co(NO.sub.3).sub.2.6H.sub.2 O 0.00028 (NH.sub.4).sub.2 Mo
O.sub.4 0.00028 EDTA (disodium salt) 0.14
______________________________________
The medium was sterilized at 15 psig steam pressure (121.degree. C.
for 15 to 20 minutes) and the pH was adjusted to 6.8 with NaOH.
To the medium, ampicillin and kanamycin were added in sufficient
amount to give a concentration of 25 mg/L for each antibiotic. The
solution of antibiotics was sterilized by filtration.
During the fermentation, three additional feedings of nutrients
were added to the fermentor. The first feeding (at an A.sub.550
=30-35) consisted of 250 g of NZ Amine A and 250 g of glycerol
dissolved in one liter of water. This allows the cell density to
increase to A.sub.550 of 50-60 before temperature induction. At
cell densities of 50-60 (23-25 hours after inoculation), the
fermentor was again fed 250 g NZ Amine A plus 250 g glycerol and
the bacteria were induced to synthesize bGH by raising the
temperature to 42.degree. C. for one hour. At an A.sub.550 =90-100,
a final feeding of 125 g NZ Amine A plus 125 g glycerol was added
so that nutrients were available for the remaining induction
period.
B. Fermentor Operation
The operating conditions that gave the best results are set forth
in this section.
1. Time Period: 0-24 Hours
a. Temperature of medium=28.degree. C.
b. Agitator speed: 1000 RPM.
c. Energy input by agitator: 0.5-1.5 horsepower per 100
gallons.
d. Aeration rate: 10 L (STP) per minute.
e. Back pressure: 3 lbs per in.sup.2.
f. Dissolved oxygen: 50% of air saturation value.
g. Additional feeding at 16 hours. A.sub.550 =30-35.
h. Absorbance of light (wavelength 550 nm) by the fermenting
medium. A.sub.550 at induction=50-60 by 24 hrs.
2. Time Period: 24-32 Hours
a. Temperature of medium.
(1) 42.degree. C. for 24-25th hours.
(2) 40.degree. C. for 25-32nd hours.
b. Agitator speed: 1200 RPM.
c. Energy input by agitator: 1.0-2.0 horsepower per 100
gallons.
d. Aeration rate: 10 L (STP) per minute.
e. Back pressure: 3-6 lbs per in.sup.2.
f. Dissolved oxygen: 10-40% of air saturation. In order to obtain
these values, the inlet air is enriched with oxygen and mixed prior
to introduction to the fermentor through the main sparger.
g. Final absorbance: A.sub.550 of 100-123.
h. Additional feedings at 24 hours and at 29 hours.
C. Results
The results obtained from three typical runs using the procedures
specified above were as follows.
______________________________________ Final Assays of Fermentation
Medium for .DELTA.9-bGH. Assay Method High Performance Liquid
Chromatography (HPLC) Bovine Growth Back Final Number of Hormone
Grams Run Pressure Absorbance Cells per per Liter No. Lbs. per
in.sup.2 A.sub.550 nm ml (Final) (HPLC)
______________________________________ 52 5 112 5 .times. 10.sup.10
3.73 53 3 99 5 .times. 10.sup.10 3.61 54 3 123 5 .times. 10.sup.10
5.93 ______________________________________
The conditions used in the foregoing fermentations in the 10-liter
fermentor were used in a pilot plant to produce larger quantities
of bovine growth hormone, .DELTA.9-bGH.
Recovery of .DELTA.9-bGH from E. coli Cells
The cells obtained from 86 liters of fermentation broth produced in
a pilot plant by procedures described above for the 10-liter
fermentor were separated from the broth by centrifugation and
resuspended in 50 liters of a buffer containing EDTA (20 mM) and
NaH.sub.2 PO.sub.4 (100 mM), adjusted to pH 7.8 with sodium
hydroxide. The cell suspension was passed through a Manton-Gaulin
homogenizer two to three passes at a pressure of 8,000 psig in
order to disrupt the cells. Intact inclusion bodies of .DELTA.9-bGH
were collected by centrifugation (13,000 g, 10 minutes) and thus
separated from cellular debris. The recovered inclusion bodies
(7,000 grams) were then washed in a buffer containing EDTA (10 mM),
and a NaH.sub.2 PO.sub.4 (0.2 M) adjusted to pH 7.5 with sodium
hydroxide. The inclusion bodies were recovered from the washing
buffer by centrifugation and dissolved in 200 liters of 8 M
guanidine hydrochloride and 60 mM ethanolamine adjusted to pH 9.0
with sodium hydroxide. The solution was stirred for 12 hours to
complete the unfolding of the pGH molecules.
Guanidine hydrochloride was removed from the solution by
diafiltration through PM-10 membranes in the form of hollow fibers.
The membranes had an average pore size of 15 .ANG. which allows
passage of molecules having molecular weights of 10,000 or less.
After all the guanidine hydrochloride had been removed from the
solution, the solution was centrifuged at 13,000 g for 10 minutes
in order to remove proteinaceous impurities and bGH molecular
aggregates which precipitated out of the solution upon removal of
the guanidine hydrochloride. The pGH was then purified by ion
exchange chromatography using a Whatman DE-52 ion exchange gel
(DEAE Cellulose) loaded into a 25 centimeter by 15 centimeter
column. The solution containing the soluble, renatured bGH was
loaded onto the column and the bGH was collected in the run through
effluent which did not bind to the column. Column fouling and
plugging, which had been observed during ion exchange
chromatography when the urea-based recovery process was employed,
was not apparent. The ion exchange chromatography step was repeated
if the desired purity was not achieved.
The solution containing the bGH was then further purified by
ultrafiltration through a PM-10 hollow fiber membrane to yield a
solution containing 0.2% bGH. Low molecular weight contaminants
were then removed by ultrafiltration against Cornell buffers. This
procedure was done a first time against 50% Cornell buffer and a
second time against 2% Cornell buffer. The solution was then
concentrated by ultrafiltration through PM-10 hollow fibers to
yield a solution containing 0.2% to 2% bGH. The solution was then
centrifuged and the supernatant was filtered through a 0.2 micron
pore filter. The bGH in the solution was then lyophilized to
produce bGH in a powdered, bioactive form.
* * * * *